In addition to their established role as cytotoxic effector cells, previous studies proposed that NK cells may regulate B cell responses to antigens. Both enhancement and suppression of Ig synthesis by NK cells have been reported, however most previous studies have utilized activated and/or cultured NK cell populations. Yuan et al. have examined mouse NK-B interactions and demonstrated that NK augmentation of B cell polyclonal Ig secretion is IFN- mediated. Their studies have also demonstrated that NK augmentation is mediated on both IgG and IgM responses. Therefore, we have evaluated the capacity of human NK and T cells to interact with, and activate, autologous resting B-lymphocytes in vitro. Using highly purified B lymphocytes the results showed that a 5 min. interaction with NK cells was sufficient to induce tyrosine phosphorylation and differentiation of B-lymphocytes into Ig-producing cells, while B cells that were not exposed to NK cells did not show these effects. These effects were specifically dependent on viable NK cells since T cells or glutaraldehyde-fixed NK cells failed to activate the B-lymphocytes. Pre-treatment of NK cells with anti-CD40L (CD154) or with CD40-mIg fusion protein blocked B-lymphocyte activation in a dose dependent fashion, while IL-2 treatment enhanced the NK-mediated effects on IgM and IgG production. The lack of expression of an antigen receptor complex on NK cells suggests that their stimulating effect on B cells is antigen-independent, and the other findings demonstrate that the mechanism does require activation by NK cells, but is dependent on close cell-cell contact. The results described above strongly suggest that components of the innate immune system (in this case NK cells) can provide an amplification/instructive role for the development of humoral immune adaptive responses. In order to investigate whether these immunoregulatory effects are physiological, we have developed a mouse model which recapitulates the findings with human cells. Using highly purified splenic B, T, and liver-associated NK cells, we have been able to show that co-culture of NK and B cells resulted in increased production of IgG and IgM in vitro, while co-culture of B cells with purified T cells had little or no effect. Specifically, co-culture of B cells with liver NK cells from either untreated mice or mice treated in vivo with IL-2 resulted in increased murine Ig (IgG and IgM) production whereas T cells failed to produce this enhancing effect. Therefore, the successful development of a mouse model which recapitulates the human NK/B effects will allow us to examine the mechanisms for this effect and extrapolate those findings to physiological primary and secondary antibody responses after challenge with different antigens. These studies will allow us to determine the role, if any, that Ly49 family members on NK cells play in the development of humoral immune responses, and may provide new insight into more effective generation of beneficial antibody responses or the role of Ly49s in the regulation of pathologic humoral immune responses. Figure 3. Effect of murine NK cells on Ig Production. Figure describes a typical experiment with mixture of NK or T cells with B cells for 4-7 days with subsequent measurement of mouse Ig levels using ELISA assay. Left panel examines mixtures of B cells with untreated or IL-2 treated liver NK cells and untreated splenic T cells. The right panel examines the NK:B ratio required for maximal enhancement.